Chikungunya virus (CHIKV; family Togaviridae; genus Alphavirus) is
an arthropod-borne virus transmitted to humans by Aedes spp. mosquitoes
(1). It is an enveloped, positive-sense, single-stranded RNA virus with
a genome of -11.8 kb (2). Three genotypes have been identified: Western
African, East Central South African (ECSA), and Asian (3).

First identified in Tanzania in the mid-1950s, CHIKV circulated in
the 1960s in sub-Saharan Africa and several Asian countries (4,5).
Reemergence of CHIKV (ECSA genotype) was reported in Democratic Republic
of Congo in 1999-2000 (6,7) and in Kenya in 2004 (4). This genotype
emerged in Comoros followed by Reunion Island, the Seychelles,
Mauritius, Mayotte, and India in 2005 (the Indian Ocean outbreak); in
Sri Lanka and Malaysia in 2006; in Singapore and Thailand in 2008; and
in China in 2010 (8).

In Cambodia, CHIKV was first detected in 1961, probably the Asian
genotype that was circulating in the region at that time (9). Since
2000, all blood specimens collected by the National Dengue Control
Program, Ministry of Health Cambodia, during hospital-based surveillance
of dengue and investigation of suspected dengue cases have been screened
for IgM against CHIKV, dengue virus (DENV), Japanese encephalitis virus
(JEV), and other arboviruses. Despite this testing, no evidence of acute
or recent CHIKV infections has been found. To confirm CHIKV infection in
samples positive by serologic testing, we conducted realtime reverse
transcription PCR (RT-PCR) and complete genome sequencing of the
samples. In 2011, we detected CHIKV ECSA genotype in patients in
Cambodia and analyzed the phylogenetic origin of the strains.

The Study

We obtained samples from 3 sources: national dengue-like
surveillance, an encephalitis study, and the outbreak investigation.
From surveillance, during 2000-2011, an average of >700 paired serum
samples were collected annually from patients admitted to sentinel
hospitals for dengue-like syndrome (Battambang, Siem Reap, Kampong Cham,
Takeo, Phnom Penh; Figure 1) in the National Dengue Control Program
(10). From the encephalitis study, conducted July 2010 through July
2011, samples from 196 patients were collected as part of a surveillance
study of central nervous system infections in Jayavarman VII hospital in
Siem Reap (with written consent from patients or legal guardians and
study approval by the National Ethics Committee in Cambodia). During the
outbreak investigation, serum was collected during investigations by
National Health authorities in Preah Vihear Province on August 16 (n =
9) and December 9-10, 2011 (n = 8), of outbreaks of suspected
measles-like or dengue-like illnesses (Table).

All serum was tested at the Institut Pasteur in Cambodia.
Acute-phase and/or convalescent-phase specimens were tested for IgM
against flaviviruses (DENV, JEV, and Langat virus) and alphaviruses
(CHIKV Ross C 347 strain and Sindbis virus). We used in-house
IgM-capture ELISA as described by Vong et al. (11) with JEV, Langat,
Sindbis, DENV, and CHIKV antigens; CHIKV was isolated by use of a
mosquito cell line (clone C6/36 of Aedes albopictus cells) (11). Viral
RNA was extracted from 140 [micro]L of serum by using the QIAamp Viral
RNA Mini Kit (QIAGEN, Hilden, Germany) according to manufacturer's
recommendations. The presence of CHIKV RNA was determined by real-time
RT-PCR selective for the E1 gene, according to a protocol adapted from
Pastorino et al. (12) for a different Taq polymerase kit (SuperScript
III Platinum One-Step Quantitative RT-PCR kit; Invitrogen, Carlsbad, CA,
USA). Each series of tests included a negative control.

[FIGURE 1 OMITTED]

Among 19 samples positive for CHIKV by real-time RT-PCR, 8 were
selected for complete genome sequencing. A total of 22 overlapped PCR
products were obtained by using primers published by Schuffenecker et
al. (13) and sent to Macrogen (Seoul, South Korea) for sequencing.

Sequence assembly and alignment were performed by using the CLC
Main Workbench 5.5 package (CLC bio A/S, Aarhus, Denmark). The complete
coding region (11,319 nt) of 8 CHIKV isolates from Cambodia were aligned
with 64 reference strains available in GenBank. Phylogenetic analysis
was performed by using the maximum-likelihood approach incorporating the
GTR+r4 model of nucleotide substitution with a bootstrap resampling
process of 1,000 replications.

During this reemergence of CHIKV in 2011, a total of 24 patients
with fever, sometimes associated with acute arthritis or encephalitis
(suggesting that many classical infections were not reported because
encephalitis is a rare complication of chikungunya), had positive RT-PCR
and/ or IgM-capture ELISA results for CHIKV. The first 2 cases were
identified in May 2011 by the National Dengue Control Program in
Battambang Province (eastern Cambodia) near the Thailand border (Figure
1). These 2 cases were in children hospitalized for suspected dengue.
Subsequent cases were reported the same year in Siem Reap (2 cases, June
and July), Kampong Thom (1 case, July), Kampong Cham (1 case, October),
and Kandal (1 case, December) Provinces. Two other outbreaks were
documented in villages in Preah Vihear Province (northern Cambodia) in
August (9 cases) and December (8 cases) 2011 (Table). The sequence of
outbreaks, in time and space, suggests that the virus was introduced to
areas bordering Thailand and progressed through Cambodia, affecting city
and villages along major northwest to southeast routes (Figure 1).
Average patient age was 20 years (range 2-56 years); cases were
distributed equally among male and female patients.

Conclusions

The alignment of the E1 gene sequence of CHIKV showed that all 8
strains carried the same amino acid substitution in the E1 protein
(E1-A226V) as did the strains that were naturally selected by the
mosquito vector a few months after the beginning of the Indian Ocean
outbreak (13). Phylogenetic analysis of the complete genome sequence
revealed that all viruses from Cambodia clustered with those isolated
during the Indian Ocean outbreak and within the ECSA phylogenetic group
(Figure 2). These isolates from Cambodia were closely related to the
viruses isolated from southern Thailand during the 2008-2009 outbreak
with bootstrap values <70 (data not shown) and to other isolates from
the recent outbreaks in Asia (Singapore, Malaysia, Indonesia, and
China). The pairwise nucleotide comparison of the complete coding region
showed a high average percentage of similarity (>99.50%) with the
recent isolates from Thailand, Malaysia, Singapore, and China. The
identity between the strains from Cambodia ranged from 99.89% to 99.93%
at the nucleotide level. Of note, the Cambodian strains can be separated
into 2 groups supported by a bootstrap value of 100, suggesting that the
viruses isolated in Battambang and Preah Vihea Provinces, which each
border Thailand, could have been introduced separately, although we
cannot exclude the possibility of introduction from other Asian
countries as well.

As numbers of reported cases, numbers of provinces affected, and
abundance of mosquito vectors (Ae. aegypti and Ae. albopictus) increase,
the risk for local transmission will probably increase in the next few
years, and levels of CHIKV infection could reach those of DENV
infection. The ECSA genotype could then become endemic to Cambodia,
which could face the same situation as in the 1960s, when a number of
chikungunya cases were reported in Cambodia, although the 1960s epidemic
did not last long (according to data available) and was not followed by
continuous virus circulation leading to successive outbreaks. As CHIKV
reemerges after 50 years of absence or low-level transmission,
cocirculation with DENV might cause substantial challenges for public
health, especially hospital overloading and increased needs for case
management. This outbreak of CHIKV ECSA genotype spread rapidly in
Cambodia over a short 7-month period. The outbreak should serve as a
warning for health authorities to prepare, not only in Cambodia, but
also in other areas where, to our knowledge, this genotype has not been
reported, such as Vietnam and Lao People's Democratic Republic.

[FIGURE 2 OMITTED]

Acknowledgments

We thank Mao Sok, Ky Santy, Yun Saophondara, Deng Ankirboss, and
Eng Chaya, and the staff of the Technical Bureau of Preah Vihear
Provincial Health Department, for providing samples, and we thank Ung
Sam An and the staff from National Institute of Public Health Cambodia
for their fruitful collaboration.

This work was supported by French Ministry of Research. The
serosurveillance studies and outbreak response were funded by a World
Health Organization grant to the Ministry of Health and Institut Pasteur
in Cambodia in 2011. The World Health Organization acknowledges the
Korea International Cooperation Agency and the World Bank for their
generous support for these activities.

Dr Duong is a senior virologist at the Institut Pasteur in
Cambodia. His research interests include arboviruses and emerging
zoonotic viruses.